Current methods for isolating nucleic acids from cultured cells generally include lysing the cells and inactivating nucleases using chaotropic salts, such as guanidine hydrochloride or guanidinium thiocyanate, and a nonionic surfactant. The released nucleic acids are then selectively precipitated from solution. The combination of chaotropic salts and nonionic surfactants, however, typically are ineffective at penetrating whole tissues to release nucleic acid.
When cells are lysed to release nucleic acids, endogenous nucleases, enzymes that degrade nucleic acids (DNA and RNA), are also released. Thus, when minimally degraded (i.e., high integrity) nucleic acid is desired, one tries to minimize nuclease activity as much as possible. Deoxyribonucleases (DNAses), which are nucleases that degrade DNA, can often be inactivated by adding divalent cation chelators to the reaction compositions. Free magnesium ions, which are typically needed for DNAse activity, are bound or complexed by the addition of chelators. Thus, chelators typically diminish or inactivate endogenous DNAses. Many endogenous ribonucleases (RNAses), nucleases that degrade RNA, however, do not typically need divalent cations, such as magnesium, for their activity. Such RNAses are unaffected by the addition of chelators. To inactivate RNAses during RNA isolation procedures, therefore, one may add to the reaction composition inhibitors of RNAse activity.
Various chemical disruption methods, using surfactants, chaotropes, proteases, bile salts, organic solvents, and harsh acidic or basic conditions have been employed to lyse cells and release nucleic acid. Proteinase K, a broad specificity protease, has also been employed. To increase the activity of Proteinase K, denaturing agents, such as anionic detergents and chaotropes have been used in combination with Proteinase K. The nucleic acid obtained using such methods may be degraded due to long incubation times or exposure to harsh conditions.
Physical disruption methods using tissue homogenizers, mortar and pestle, dounce homogenization, frozen pulverization, and bead beaters, have been employed with biological samples. The nucleic acid obtained using these methods, however, is typically degraded due to shearing, long incubation times and high temperatures that may be generated using these methods. Further, such methods present health and safety concerns as a portion of the sample may be aerosolized during the physical disruption procedure. This becomes a critical issue when the sample potentially contains infectious agents such as HIV, hepatitis, or other pathogenic microorganisms.
Thus, there is a need for methods and compositions for isolating high integrity, i.e., high molecular weight, nucleic acid molecules from biological samples in a safe, rapid, and efficient manner, especially from whole tissues.